Production of “biological carriers” for induction of immune responses and inhibition of viral replication

ABSTRACT

This application provides a method to form non-infectious Biological Carrier that may be used to deliver signals to cells either in vitro or in vivo. The Biological Carriers are inactivated virus particles that have been specifically modified to give biological properties different from the virus particles deriving from an unmodified host cell that (i) expresses at least one co-stimulatory molecule and (iia) at least one antigen that can initiate an immune response, and/or (iib) express surface molecules that suppress viral replication.

PRIORITY DOCUMENTS

This application is a 371 application of PCT/US02/04157, filed Feb. 12,2002, which claims priority to U.S. Provisional application 60/268,066,filed Feb. 13, 2001.

FIELD OF THE INVENTION

This invention relates to the field of antigen presentation for theactivation and resultant induction of antigen-specific immune responsesand the inhibition of viral replication by the formation of “BiologicalCarriers.”

BACKGROUND OF THE INVENTION

In mammals, antigen-presenting cells (APCs) process foreign antigens.The processing of the antigen within the APC triggers an efficientimmune response within the host. Antigens are degraded into peptidefragments and become bound to major histocompatibility complex (MHC)molecules that are expressed on the cell surface and are able tointeract with other cells of the immune system. Dendritic cells,macrophages, and Kupffer cells in the liver are among the most commonlyencountered type of APCs. These cells readily engulf foreign particlesand express MHC molecules on their plasma membrane surface. These MHCsurface molecules, know as human leukocyte antigens (HLA) in humans, areinvolved in the immune response against disease.

There are two classes of MHC molecules (class I and class II). MHC classI molecules display exogenous antigens (i.e., antigens taken up fromoutside the cell), whereas MHC class II molecules display endogenousantigens (i.e., antigens that originate within the cell) to the immunesystem. Processing of the antigens differ in each case. Processing ofexogenous antigens by APCs occurs in stages. The cells first take up theantigens by endocytosis. The internalized endocytic vesicles then fusewith lysosomes where the foreign antigens are hydrolyzed by lysosomalenzymes, resulting in peptide fragments of 10 to 20 amino acids. Thesepeptide fragments bind to a cleft within the MHC molecule and aretransported to the cell's surface for interactions with cells of theimmune system. The processing that leads to the display of endogenousantigens can arise from viral infection, etc. Such antigens are cleavedin the cellular cytosol, and transported into the lumen of theendothelial reticulum, where they become associated with MHC class Imolecules, which are then transported to the cell surface. Foreignantigens can promote an immune response, whereas peptide fragmentsderived from cellular proteins when bound to MHC class I molecules andpresented on the cell's surface are recognized as “self” and will notusually elicit an immune response.

Processed antigens displayed on self-MHC molecules supply one of the twosignals required for T-lymphocyte activation. In addition to recognitionof a foreign antigen fragment, simultaneous delivery of a co-stimulatorysignal is needed for the activation of naïve T-lymphocytes. Theseco-stimulatory signals together with both class I and class II MHCmolecules are molecules present on professional APCs. The presence ofthese molecules on professional APCs stimulate the clonal expansion ofnaïve T-lymphocytes, resulting in their differentiation into armedimmune effector cells, and ultimately memory cells. Priming is a processwhere naïve T-lymphocytes are activated by the first time exposure to anantigen, whereas re-exposure to foreign antigens result in activation ofmemory cells.

Activation occurs when the T-lymphocyte's T cell receptor (anantigen-specific receptor) and its co-receptors (either CD4 or CD8molecules) recognize the foreign peptide-MHC complex, simultaneouslywith a co-stimulatory signal delivered from the same APC. Thebest-characterized co-stimulatory molecules on APCs are CD80 (B7-1) andCD86 (B7-2). These structurally related molecules are members of theimmunoglobulin superfamily and recognize the CD28 molecule onT-lymphocytes, resulting in T-lymphocyte activation. Activation ofT-lymphocytes is controlled by the subsequent expression of CTLA-4. TheCTLA-4 receptor is closely related to the CD28 molecule that binds theB7 molecules with a higher affinity than CD28 and prevents furtherT-lymphocyte activation.

There are numerous examples of how the addition of co-stimulatorymolecules to cells affects cellular processes. In HIV/AIDS research:When CD3 and CD28 receptors on cultured T-lymphocytes are stimulated byimmobilized monoclonal antibodies (mAbs), expansion of polyclonal CD4positive T-lymphocytes occur. Ifthe T-lymphocytes were obtained fromFIV-infected donors, HIV-1 viral load declines (in the absence ofantiretroviral agents) simultaneously with T-lymphocyte expansion.Moreover, CD28 stimulation rendered these cells highly resistant toHIV-1 infection, mediating an antiviral effect early in the viral lifecycle before HIV-1 DNA integration. The HIV-1 resistant state isspecific for the macrophage-tropic HIV-1 isolates and is due to the lackof CCR5 receptor transcription, which is a required secondary receptorfor HIV-1 macrophage-tropic virus infection. In tumor biology: Theintroduction of either MHC class II molecules or co-stimulatorymolecules to tumor cells results in their efficient rejection in vivo.In virology: Viral infection with a number of the herpesviruses causes adiminution of cell surface co-stimulatory molecule expression, resultingin viral replication without mounting any immune response towards theinfected cells.

Many viruses produce degenerative changes in cells when replicating in asusceptible cell culture. These characteristic changes are calledcytopathic effects and are associated with certain morphologic changesin the host cell. The intracellular sites where the events of viralreplication take place vary among the viral families. Enveloped virusesmature by a budding process, although some budding occurs withnon-envelope containing viruses. For envelope viruses, viral-specificenvelope glycoproteins are inserted into cellular membrances and theviral nucleocapsids then bud through the membrane at these modifiedsites. In this process, the virus acquires their envelope forinfectivity and can also acquire cellular-related molecules. Studieswith HIV, Influenza, and Chlamydia have shown virus particles that haveincorporated HLA molecules into the mature virus particle. During theinfection the cell is destroyed and the virus particles are releasedinto the culture supernatant. The amount of infectious virus present inthe cell culture fluid can be titrated and infectivity inactivated by avariety of methods. Although inactivated virus particles have lost theability to replicate they maintain their structure, as detailed in thisapplication, they can be used as a scaffold to carry cell surfaceexpressed molecules.

SUMMARY OF THE INVENTION

This invention provides for the formation of non-infectious BiologicalCarrier to deliver signals to cells either in vitro or in vivo (see FIG.1A). The Biological Carriers are whole virus particles that are eithertotally inactivated (by biological, chemical, genetic or mechanicalmeans), or partially inactivated for subsequent viral infection. Theyare produced in cells that are genetically modified to over-expresssurface molecules that are able to elicit immune responses (see FIG. 1Bfor concept in schematic form). The invention is intended for in vivouse in any recipient where enhancement of immune responses can beadvantageous to that individual, although in vitro pretreatment of cellscan also be envisioned.

Viruses are ideal candidates for utilization as non-professional antigenpresenting carriers. The invention uses viruses not for their infectiousabilities (all preparations will be inactivated), but as a scaffold thatcontain specific antigens and co-stimulatory molecules to induce immuneresponses. The major advantage of this approach is the ease ofproduction of potential therapeutic and/or vaccine doses. At the end ofthe virus life cycle, large amounts of virions are released frominfected cells, reaching concentrations of 10¹² virus particles permilliliter of culture fluid. Intrinsic to virus release, portion of thecell membrane are removed as the mature virus particle buds from thecell. The cells that support the productive viral infection can begenetically engineered to over-express surface molecules that would becarried with the virus particle as it is released from the cell. Inaddition, MHC class I and class II molecules containing viral peptides(due to the active viral infection) will decorate the virus particles,thereby presenting the antigens needed to stimulate the CD3 receptor onT-lymphocytes. These interactions supply one of the two signals requiredfor T-cell stimulation. The over expression of co-stimulatory moleculesby genetic engineering supplies the second signal, leading to antigenspecific immune stimulation. These are distinct advantages over the useof cells (professional or non-professional) to present antigens toT-lymphocytes. The current procedure with professional antigenpresenting cells (APCs) involves isolating cells from a patient, growingthem in culture, and transplanting them back into that same person, aprocess that takes weeks. With non-professional APCs, a geneticengineering step (where co-stimulatory and/or MHC molecules would beintroduced into the cell) would be added to the process. Even if thecells can be implanted allogeneically, current procedures arecumbersome, labor intensive, time consuming, and expensive. The presentinvention simplifies the process, thereby making immunotherapypotentially available world-wide in the area of infectious diseases.

In one aspect, the invention provides a Biological Carrier preparationthat (i) contains an antigen (here, the antigen can be a protein,polypeptide, lipid or glycolipid) and/or antigen fragment bound to aprimary surface molecule of said host cell such that the BiologicalCarrier contains at least one antigen fragment presented to initiate animmune response and (ii) at least one co-stimulatory molecule. In oneembodiment of this aspect, the Biological Carrier preparation isvirus-specific. That is, a specific virus is grown in a fully permissivecell line and while budding from the cells' surface contains at leastone antigen specific to that virus processed into an antigen fragment.The Biological Carrier preparation can be prepared by infection of apermissive host cell line (or primary cell), from a chronically infectedcell line, from a packaging cell line (a cell engineered to express avirus particle capable of one replicative infectious cycle), or fromcells isolated directly from the mammal (cells isolated from the tumoror of non-tumor source). In addition, the Biological Carrier preparationcan be from the native harvested culture fluid, or the preparation canbe concentrated (e.g. centrifugation, polyethylene glycol-precipitation,or the like) and/or lyophilized for ease of storage and stability. Inanother embodiment, the Biological Carrier preparation also contains anon-specific immune stimulatory activity. That is, molecules areexpressed at the cell's surface that would indiscriminately stimulateT-lymphocytes. Such molecules can be a stimulatory antibody directedagainst the T-lymphocyte CD3 molecule, but not limited to this molecule,and when the virus buds from the infected viral cells' surface itcontains in addition to at least one viral specific antigen processedinto an antigen fragment, also a non-specific molecule that can enhanceimmune responses in the recipient. Preferably, these viral-specificantigens are in a form available for presentation. Further, theBiological Carrier preparation contains at least one co-stimulatorymolecule. Preferably the co-stimulatory molecule is cell surfaceassociated and in a form available for presentation. The primary surfacemolecules for antigen presentation are preferably MHC I, MHC II or CD1.The co-stimulatory molecule is preferably selected from the groupconsisting of CD80 and CD86, but not restricted to this group ofmolecules.

In another aspect, the invention provides a method for stimulating thepresentation of at least one exogenous antigen fragment on theBiological Carrier, which method comprises contacting a virally infectedcell that is capable of expressing at least one co-stimulatory moleculealong with (i) an endogenous viral antigen (one intrinsic to the viralinfection), (ii) an exogenous antigen (one important in initiating animmune response), (iii) an immune dominate peptide (one loaded onto theMHC molecule before or during virus expression). This exogenous antigencan be included in vitro in the culture media where it would be taken upby the virus host cell or can be supplied as genetic material that codesfor the exogenous antigen, which the cell processes intracellularly intoat least one antigen molecule or fragment. The method can furtherinclude contacting the virally infected cell with cytokines (such astumor necrosis factor-alpha) or by contacting with other reagents(phorbol esters), during the production of the Biological Carriers inorder to enhance, stimulate or induce virus expression.

In another aspect, the invention provides a method of activating orpriming a naïve T-lymphocyte to respond to viral antigens, by contactingthe T-lymphocyte with a Biological Carrier preparation containing eitherviral-specific antigen(s) fragments or with non-specific immuneactivators in the proper conformation for presentation. In addition, theBiological. Carrier may contain at least one co-stimulatory molecule.This T-cell priming can allow for the induction and expansion of notonly effector cells but also long-lasting memory T-lymphocytes against aspecific virus.

In another aspect the invention provides a Biological Carrierpreparation, which expresses at least one membrane bound exogenousantigen and/or antigen fragment and also expresses a co-stimulatorymolecule. Thus, the Biological Carrier preparations contain the antigenand/or antigen fragment available to “professional” APCs for theirprocessing and presentation to T-lymphocyte. In one embodiment of thisaspect, the Biological Carrier preparation has been contacted with atleast one antigen fragment (either exogenous or endogenous), which theviral infected cell displays on its cell surface. In another embodiment,the Biological Carrier contains at least one antigen fragment obtainedfrom the introduction of exogenous genetic material into the viral hostcell. Preferably this exogenous genetic material is in an expressionvector. Further, the Biological Carrier preparation also containsexogenous genetic material that codes for at least one co-stimulatorymolecule. Preferably, this exogenous genetic material is also in anexpression vector.

In another aspect, the invention further provides a method fordetermining the immune competence or state of activation of theT-lymphocyte population of a mammalian host to a particular class ofantigens by contacting the T-lymphocyte population with a specificbiological preparation and observing for any change in the state ofactivation (e.g. Alamar-Blue fluorescence, T-lymphocyte production ofgamma interferon, or expression of T-lymphocyte surface activationmarkers).

The present invention further relates to the treatment or prevention ofa disease in a mammal, which may be a human or non-human, byadministering to the mammal a Biological Carrier preparation that (i)contains at least one viral-specific antigen bound to a primary surfacemolecule that can be presented to initiate an immune response and (ii)also contain at least one co-stimulatory molecule (e.g., B7-1 and/orB7-2). In one embodiment of this aspect of the present invention, theBiological Carrier contains at least one processed viral antigen. Inanother embodiment, the Biological Carrier preparation contains at leastone processed, but exogenously added viral antigen. Such mechanisms ofprevention and/or treatment in mammals have application with respect tovarious diseases (whether viral, bacterial, fungal, or other origin),cancer, toxin exposure (whether viral, bacterial, fungal, or otherorigin), or antigens of plant origin (e.g. poison ivy, poison sumac),and the like.

In summary, the Biological Carrier preparation system has a wide rangeof applications, including but not limited to, in vitro or in vivoactivation and expansion of antigen specific T-lymphocytes for use inadaptive cellular immunotherapy against infectious diseases and cancer,for use of Biological Carrier preparations for vaccines and/orimmunotherapeutics, and for an in vitro assay system for determining anindividual's immune potential or potentiation to any antigenic epitopes.The present invention provides a method to form non-infectiousBiological Carrier to deliver signals to cells either in vitro or invivo. The Biological Carriers of the present invention are inactivatedvirus particles that have been specifically modified to exhibitbiological properties that differ from those of virus particles derivingfrom an unmodified host cell. The modified cell that is host to thevirus (i) expresses at least one co-stimulatory molecule and (iia) atleast one antigen that can initiate an immune response, and/or (iib)express surface molecules that suppress viral replication. Theco-stimulatory molecule is one of a class of molecules that either aloneor in combination with other molecules is capable of T-lymphocytestimulation leading to specific antibody formation and/or cytotoxicT-cell activity in the host. Specificity is conferred to the BiologicalCarrier by the presence of endogenous and exogenous processed antigensthat are bound to Class I and Class II major histocompatibility complex(MHC) surface molecules that are present on the modified cell. Processedantigens can be intrinsic to the infectious process or disease specificantigens from said host cell. Also disclosed is a method for inhibitingviral replication by stimulating cells with Biological Carrierscontaining molecules that stimulate specific receptors that are presenton human peripheral blood mononuclear cells that are able to preventviral entry. Thus, disclosed are methods for the treatment or preventionof a disease in mammals including humans.

The present invention particularly concerns:

1. A method for inducing an antigen-specific T-cell response in a mammalcomprising administering to the mammal an effective amount of aBiological Carrier preparation that has been derived from a host cellthat contained at least one fragment of said antigen bound to a primarysurface molecule of said host cell, and which also expressed at leastone co-stimulatory molecule, said molecules being carried on theBiological Carrier preparation and inducing an immune response againstthe specific antigens that were processed in said host cell.

2. A method to inhibit viral replication and/or viral entry in a mammalcomprising administering to the mammal an effective amount of aBiological Carrier preparation that has been derived from a host cellthat contained at least one molecule either alone or in combination withone or more other molecules expressed on said host cell, said host cellexpressing one or more molecules that prevent expression of moleculesrequired for viral entry, said molecules required for viral entry beingcarried on the Biological Carrier preparation leading to an inhibitionof viral replication.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further described by the accompanying drawings and thedescription thereof herein, although neither is a limitation of thescope of the invention.

FIG. 1A is an illustration of the interaction of the Biological Carrierpreparations with dormant T-lymphocyte populations, resulting in theiractivation and induction of specific immune responses; FIG. 1B)schematically represents formation of the carrier particles.

FIG. 2 shows that when Biological Carriers (either HSV-2 or HIV-1 based)are produced from untransduced host cells the degree of relativefluorescence (a measure of lymphocyte activation) is similar tounstimulated cultures. PHA-stimulated cultures were included to showthat the cells were capable of stimulation as measured by theAlamar-Blue fluorescent assay. Values were from day 6 cultures.

FIG. 3, Panel A shows the stimulatory ability at day 6 of HSV-2 basedBiological Carriers prepared from anti-CD3+B7's transduced cultures(A+B) to stimulate T-lymphocytes compared to HSV-2 based BiologicalCarriers prepared from untransduced cultures (Un) in three differentdonor lymphocyte preparations. FIG. 3, Panel B shows the comparativestimulatory activity of HSV-2 based Biological Carriers prepared fromuntransduced (Un), anti-CD3 (A), B7-1+B7-2 (B), and anti-CD3+B7's (A+B)on elutriated peripheral blood lymphocytes after 14 days in culture.

FIG. 4 shows the stimulatory ability at day 6 of HIV-1 based BiologicalCarriers prepared from either B7-1+B7-2 (B−) or anti-CD3+B7's (A+B)transduced cultures to stimulate lymphocytes compared to HIV-1 basedBiological Carriers prepared from untransduced cultures (Un) in threedifferent donor peripheral blood lymphocyte preparations.

FIG. 5 shows the ability of lyophilized HSV-2 based Biological Carrierpreparations from anti-CD3 (A), B7-1+B7-2 (B), and anti-CD3+B7's (A+B)transduced cultures to stimulate lymphocytes compared to the samepreparation that was not lyophilized after 13 days in culture. Theenhanced stimulation in the case of the lyophilized preparation isprobably due more to the larger volume (5 mL) of native culturesupernatant lyophilized and tested in the experiment than to thelyophilized procedure itself.

FIG. 6 shows that the concentration of the native culture supernatant(by polyethylene glycol precipitation) maintains the stimulatoryactivity of the HSV-2 based Biological Carriers obtained fromanti-CD3+B7's transduced cultures. This was a 40-fold concentration,allowing smaller volumes (25 μL rather than 1 mL) of the preparation tobe used to obtain similar effects. The time point shown is 10 days afterthe addition of Biological Carriers.

FIG. 7 shows that the lymphocyte response can be re-activated in thesame cultures that where initial stimulated even after the lymphocyteswere rested for over one month in culture. The Figure shows twoexperiments with two different donors. Experiment 1 used native culturesupernatants for the initial stimulation and re-stimulation; experiment2 used polyethylene glycol precipitated culture supernatants for theinitial stimulation and re-stimulation. Note that the initialstimulation in experiment 2 is the same as the experiment shown in FIG.6.

FIG. 8 shows the increase in HIV-1 encoded p24 antigen expression afterexposure of elutriated T-lymphocytes to either HIV-MN (alymphocytotropic HIV strain) or HIV-BaL (a monocytotropic HIV-1 strain).Although each HIV strain requires a different secondary receptor forcellular entry (CCR5 for monocytotropic strains and CXCR4 forlymphocytotropic strains) the viral strains used are replicationcompetent in these cells. This experiment used Donor #9 elutriatedlymphocytes.

FIG. 9 shows the effects of exposing elutriated T-lymphocytes to HIV-1based Biological Carrier preparations. Unlike preparations made fromuntransduced host cells, the Biological Carrier preparations made fromhost cells modified to express either B7-1+B7-2 or the B7 molecules+ananti CD3 molecule resulted in a dramatic decrease in the ability ofHIV-1 (either lymphocytotropic or monocytotropic) to replicate in humanperipheral blood T-lymphocytes. This experiment used Donor #9 elutriatedlymphocytes.

FIG. 10 shows the effects of exposing elutriated lymphocytes to HSV-2based Biological Carrier preparations. Unlike the HIV-1 based BiologicalCarrier treatment shown in FIG. 9, HSV-2 based Biological Carriertreatment did not inhibit HIV-1 replication. This data attests to thespecificity of the Biological Carrier preparations. The preparationsshow specificity only towards the virus used to prepare the BiologicalCarrier preparation. This experiment used Donor #9 elutriatedlymphocytes and was done at the same time as the experiment in FIG. 9.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to the expression of surface molecules onconstructed cell lines that are host to specific viruses or viral-likeparticles. The virus may contain either a DNA or RNA genome, or becomposed of material that induces a budding process from cells. Theexpressed surface molecules can be endogenous to the cell line selected,or can be specific for one or more molecules expressed on the surface ofa given cell by biological, chemical, or mechanical means. The surfacemolecule can be naturally expressed in nature on a cell's surface, orcan be engineered as such by molecular, chemical, or mechanical means.The cell lines can be chronically-infected with the virus or the viruscan be introduced into the cell by biological, chemical, or mechanicalmeans. The formation of the budding particle containing native (thosesurface molecules naturally present on said cell surface) and/orspecific molecules (those surface molecules that were intentionallyintroduced on said cell surface) is used as a carrier (referred toherein as a “Biological Carrier”) of that material for the purpose ofsignaling or modifying specific cellular events.

The present invention relates to, but is not limited to, antigenpresentation leading to the activation of immune responses. Immuneresponses can be specific expansion or activation of one or more cellpopulations. The “Biological Carriers” can behave as antigen-presentingcells for the activation of T cell responses, or as an in vitro methodfor assessing immune responsiveness to specific infectious diseaseagents. The present invention provides for use of “Biological Carriers”to present relevant antigens and appropriate co-stimulatory molecules asan immunoprophylactic, immunotherapeutic, or vaccine candidate to treat,for example, infectious diseases, cancer, exposure to toxins, and as analternative to conventional drug and/or antibiotic therapies on whichhost resistance has developed. Pursuant of the present invention, bothHSV-2 and HIV-1 were chosen as an example of a DNA and RNA virus,respectively. However, any virus including but not limited to orinducing a budding process that incorporates membrane fragments into ascaffold particle can be used to generate a Biological Carrier. EachBiological Carrier preparation was prepared from untransduced,anti-CD3-transduced, or B7-1+B7-2 transduced with and without anti-CD3and data was presented to demonstrate the ability of the transducedcells to activate T-lymphocytes. Although the transduction of specificsurface molecule expression may generally be desired, in some cases,whether it is due to the cells selected or to the virus being used,appropriate molecules or nuances related to the viral life cycle mayeliminate the need for virus host cell surface modifications. Theinvention is further envisioned as a general way of delivering moleculesto humans in vivo. The forced surface expression of molecules that arenormally secreted or sequestered internally within cells, when expressedin the context of a “Biological Carrier” is anticipated to displayincreased stability from degradation resulting in longer and/or enhancedbiological activity. Genetic engineering of molecules for surfaceexpression and ultimately displayed on the surface of the “BiologicalCarrier” can include (in addition to molecules that would interact withcellular receptors on the responding cell) molecules whose mode ofaction require entry internal to the cell Internalization can bereceptor-mediated or mediated through biological or chemicalmodifications that allow passage across the cell's outer and/or nuclearmembranes. In the present embodiment, all “Biological Carrier”preparations will be inactivated thereby not allowing for viralreplication. However, in some instances partially inactivated, ornon-inactivated, preparations might be envisioned.

The “Biological Carriers” described herein establish an ideal system forassessing the ability of human patients to respond immunologically bytesting their T-lymphocyte responses. By assessing an individual'simmune competency, the ability to respond to a particular vaccine can bedetermined, in addition the ability of an individual can be prescreenedto be responsive to a specific “Biological Carrier” preparation beforereceiving the material in order to determine the potential benefit ofthe administration. The potent accessory cell function of the“Biological Carriers” may be able in vivo to present infectious diseaseagents and/or tumor antigens to T-lymphocytes obtained from afflictedindividuals, whose immune response apparently is inadequate to mount aneffective response to eliminate the infectious agent or tumor. Inaddition to the in vivo expansion of effective T-lymphocytes, activatedT-lymphocytes can be expanded in vitro for use in immunotherapeuticapplications. Tumor cells isolated from patients or establishedtumor-derived cell lines can be used as host for virus infections. Thevirus used in this manner can be related to the tumor in question or canbe from, or be derived from, a separate group of viruses that arepermissive to grow in said tumor cells for the expressed purpose ofbudding and thereby removing tumor specific antigens already processedin the proper configuration for T-lymphocyte presentation. These tumorcells can be in addition modified on their cell surface withco-stimulatory molecules or other accessory molecules that wouldfacilitate the “Biological Carrier's” ability to mount an immuneresponse against the tumor.

Infectious disease agent against which the present invention may beapplicable in the induction of an immune response include but are notlimited to bacteria, parasites, fungi, and viruses. The multitudes ofantigens encoded by these agents that may be processed and presented bythe “Biological Carriers” include but are not limited to externalsurface proteins and structural proteins including intracellularenzymes, transcription factors, and other cell regulatory proteins. Forexample, antigens encoded by any genes of the HIV-1 genome includinggag, pol, vif vpu, tat, rev, env, and nef may be all present as eitherintact antigens or processed and configured within the MHC molecule aspart of the “Biological Carrier” for either presentation of the intactantigens to “professional” antigen presenting cells (macrophages,dendritic cells, etc.) or directly to T-lymphocytes, respectively. Inaddition, a variety of other infectious agents including hepatitis B,hepatitis C, herpes simplex virus, varicella zoster, Epstein-Barr virus,cytomegalovirus, human herpesvirus-6, -7, -8, HIV-1, HIV-2, HTLV-1,HTLV-2, Rubella, Rubeola, Influenza, and species of Chlamydia,Helicobacter, Neisseria, Mycobacteria (especially M. tuberculosi) andToxoplasma are encompassed within the scope of the invention. Theantigen(s) can be present on the host cell either as part of theinfectious processed, naturally native to the cell, or introduced bypinocytotic uptake, or by biological (viral vectors), chemical(liposomes), or mechanical (electroporation) methods.

The following examples further illustrate experiments using BiologicalCarrier preparations that have demonstrated reduction to practice andutility of selected preferred embodiments of the present invention,although they are in no way a limitation of the teachings or disclosureof the present invention as set forth herein.

EXAMPLE 1 Requirement for Host Cell Modification(S) for BiologicalCarrier-Dependent Lymphocyte Stimulation

The principle of this invention is demonstrated by proliferationexperiments comparing the degree of stimulation of biological carrierpreparations obtained from untransduced, anti-CD3, B7-1+B7-2, andanti-CD3&B7-1+B7-2 transduced cultures.

Elutriated lymphocytes that were treated for six day with a preparationof biological carriers obtained from untransduced cultures showed adegree of proliferation (lymphocyte stimulation) similar to unstimulated(un-treated) cells (FIG. 2). Proliferation was measured by AlamarBlue™assay. The assay is designed to measure quantitatively the proliferationof human cells by the incorporation of an oxidation-reduction indicatorthat fluoresces in response to chemical reduction of growth mediumresulting from cell growth. The lack of biological carrier dependentstimulation when biological carriers were prepared from untransducedcultures was independent of the virus used to form the biologicalcarrier particles. Both HSV-2 and HIV-1 based biological carriersprepared from untransduced host cells failed to stimulate the lymphocyteproliferation. The host cell for the HSV-2 based biological carriers areLof(11-10) cells (an SV40 T-antigen transformed stromal cell line thatwas infected with HSV-2), whereas the host cells for the HIV-1 basedbiological carriers are 1119ERC cells (a chronic HIV-1 infected cellline established by the electroporation of pHXB2 that contains the HIVgenome into A3.01 cells). The introduction of genetic material codingfor either or both anti-CD3 and B7-1+B7-2 was accomplished by MuLV-basedretroviral transduction and selection of the cells that incorporated andexpressed said molecules. The data from PHA stimulated cultures wasincluded in FIG. 2 to show that the elutriated lymphocytes were capableof fluoresces in response to known proliferating compounds. However,HSV-2 based biological carriers prepared from either anti-CD3&B7-1+B7-2transduced cells in three different donor's lymphocytes (FIG. 3, panelA) or anti-CD3, B7-1+B7-2, and anti-CD3&B7-1+B7-2 transduced cells in afourth donor's lymphocytes (FIG. 3, panel B) was able to stimulateT-lymphocyte proliferation. T-lymphocyte stimulation was also observedin three different donor's T-lymphocyte populations when exposed toHIV-1 based biological carriers obtained from B7-1+B7-2 andanti-CD3&B7-1+B7-2 transduced cells, but not from untransduced cultures(FIG. 4). The identity of the donor T-lymphocytes and the key for theabbreviations used in the Figures are listed in Table 1.

EXAMPLE 2 Biological Carrier Preparations Retain Their BiologicalActivity After Lyophilization and Storage at Room Temperature

The principle of this invention is further demonstrated by retention ofcellular proliferating biological activity when biological carrierpreparations were lyophilized and stored at ambient temperatures.

The ability of native harvested culture fluid from HSV-2 basedbiological carriers from anti-CD3, B7-1+B7-2, and anti-CD3&B7-1+B7-2transduced cells were compared to aliquots of the same sample fluidsafter lyophilization with respect to the ability of the preparations tostimulate T-lymphocytes in culture. In addition to lyophilization, thelyophilized material was stored at room temperature for two weeks beforetesting biological activity. After thirteen days in culture with Donor#6 T-lymphocytes (Table 1), the lyophilized culture supernatant from thetransduced host cells showed similar stimulation of proliferation tothat observed with the native culture supernatants (FIG. 5).

TABLE 1 List of Donor Cells and Key for Biological Carrier PreparationsDonors: 1 = Lot#0G0002 Elutriated lymphocytes 2 = Lot#0G0008 Elutriatedlymphocytes 3 = Lot#0H0005 Elutriated lymphocytes 4 = Lot#0J0009Peripheral Blood Mononuclear Cells: 5 = Lot#0J0019 Elutriatedlymphocytes 6 = Lot#0H0015 Elutriated lymphocytes 7 = Lot#0H0014Elutriated lymphocytes 8 = Lot#0H0027 Elutriated lymphocytes 9 =Lot#1A0008 Elutriated lymphocytes Biological Carrier Preparations: Un(Untransduced Cultures); B (B7-1 + B7-2 transduced); A (Anti-CD3transduced) A + B (AntiCD3 + B7's Transduced Cultures)

Table 1 lists the nine different donors cells used in the data presentedin the following Figures. Eight of the nine cell preparations wereobtained by elutriation of human peripheral blood mononuclear cells andare depleted of monocytes. Donor #4 consists of ficoll-fractionatedperipheral blood. In addition, Table 1 gives the meaning for theabbreviations Un, A, B, and A+B that refer to the host cell used toprepare the Biological Carrier preparations.

EXAMPLE 3 Biological Carrier Preparations Retain Their BiologicalActivity After Concentration

The principle of this invention is further demonstrated by retention ofcellular proliferating biological activity when biological carrierpreparations were concentrated.

The ability to stimulate T-lymphocytes with native harvested culturefluid from HSV-2 based biological carriers obtained from untransduced oranti-CD3&B7-1+B7-2 transduced cells were compared to the samesupernatants concentrated by polyethylene glycol (PEG) precipitation.The addition of PEG to culture fluid results in the formation of aprecipitate. Virus (HSV-2) infected harvested culture supernatants werecentrifuged at 4,000 times the force of gravity for 10 minutes, removinglarge particulate material from the culture fluid. Polyethylene glycolwas added to the clarified supernatant to 6% and after 4 to 16 hours ofincubation at 4° C. a precipitate was collected by centrifugation.Following resuspension of the pellet (40× concentrate), the material wascompared to the native culture supernatant in T-lymphocyte proliferationassay. The PEG biological carrier material from transduced culturesstimulated T-lymphocyte proliferation similar to the unprocessedbiological carrier preparations (FIG. 6).

EXAMPLE 4 T-Lymphocytes Stimulated with Biological Carrier Preparationscan Undergo a Second Stimulation when Re-Exposed to the Same BiologicalCarrier Preparation

The principle of this invention is further demonstrated by observingsecondary responses to re-administering the biological carrierpreparation to the same population of cells.

Two donor lymphocytes, #8 and #7 in experiment #1 and #2, respectively,were initially stimulated with untransduced and transduced (anti-CD3,B7-1+B7-2, and anti-CD3&B7-1+B7-2) HSV-2 based biological carrierpreparations (FIG. 7). The data shown for the initial stimulation was 7days after exposure of the cells to the biological carrier preparation.By 14 days there was no observed fluorescent activity over theuntransduced cultures (data not shown). These cultures were kept in thisresting state for 32 days in experiment #1 and 31 days in experiment #2.After which the cultures were re-exposed to the same biological carrierpreparation used in the initial stimulation. The ability of the culturesto show a proliferative response to re-administration of the biologicalcarrier preparation suggests that the biological carrier preparationscan be used therapeutically to control and maintain immune responses.

EXAMPLE 5 Inhibition Of HIV-1 Replication Using HIV-1 Based BiologicalCarrier Preparations

The principle of this invention is further demonstrated by experimentsusing HIV-1 based Biological Carriers to inhibit HIV-1 replication. Fourday PHA/IL-2 stimulated elutriated lymphocytes support HIV-1 (both HIV-1MN and BaL) replication as measured by detection of HIV-1 encoded p24protein released into the culture supernatant over time (FIG. 8). Theaddition of HIV-1 based Biological Carrier preparation obtained fromuntransduced cultures (FIG. 9) showed similar p24 values to theuntreated cultures shown in FIG. 8. However, the addition of HIV-1 basedBiological Carriers prepared from either the B7-1+B7-2 or theanti-CD3&B7-1+B7-2 transduced cultures inhibited HIV-1 replication (FIG.9). The degree of inhibition differed in the two preparations; theanti-CD3&B7-1+B7-2 preparation showing the most significant inhibition.The specificity of HIV-1 inhibition to only the HIV-1 based BiologicalCarriers formed from either B7-1+B7-2 or anti-CD3&B7-1+B7-2 transducedcultures is further demonstrated when HSV-2 based Biological Carrierpreparations from either untransduced or anti-CD3&B7-1+B7-2 cultures didnot inhibit HIV-1 replication in the same experiment (FIG. 10). Theinhibition of HIV-1 replication is not due to the lack of lymphocyteactivation. In fact, cultures treated with either the HIV-1 or HSV-2based Biological Carriers prepared from B7-1+B7-2 transduced culturesshow higher stimulation at earlier times (day 4 for HIV and day 6 forHSV-2) than cultures treated with PHA/IL-2 alone (in the absence ofBiological Carriers).

EXAMPLE 6 HSV-1 & -2 Specific Antibody Reactivity Induced by Exposure ofPeripheral Blood Lymphocytes to HSV-2 Based Biological CarrierPreparations

The principle of this invention is further demonstrated by experimentsusing HSV-2 based Biological Carriers to induce HSV-1 & -2 specificantibody reactivity. Unstimulated peripheral blood elutriatedlymphocytes were exposed to either PHA, HSV-2 based or HIV-1 basedBiological Barrier preparations (Table 2). After 3, 6, 10 and 14 days inculture, 200 μL aliquot of the cell suspension was placed into fourdifferent wells within a 96-well culture plate. Each of the four wellswere coated with a lysate from either herpesvirus type-1 (HSV-1),herpesvirus type-2 (HSV-2), human immunodeficiency virus type 1 (HIV-1),or vesicular stomatitis virus (VSV). The cultures were incubated at 37°C. for 3 days, followed by incubation with a hydrogen peroxidaseconjugated anti-human IgG antibody and colorimetric substrate fordetection of antibodies formed in vitro against the different viruses.The results illustrate the ability of HSV-2 based Biological Carriers,but not HIV-1 based Biological Carriers, to induce HSV-1 & -2 specificreactivity. If neutralizing in nature, this antibody specific responsecan inhibit HSV-2 reactivation in vivo.

Table 2 shows specific antibody reactivity against HSV-1 & -2 whenperipheral blood lymphocytes were exposed to HSV-2 based BiologicalCarriers (BCs), but not when exposed to HIV-1 based BCs. Donor #9 cellswere used in this experiment; donor's plasma was positive for thepresence of HSV-1 & -2 antibodies at the time of lymphocyte isolation.The data shows some reactivity at day 10 in PHA-stimulated cultures. Inthis donor HSV-2 based Biological Carriers prepared from untransducedhost cells were HSV-1 & -2 antibody reactive. We would expect that in aHSV-1 & -2 negative donor only Biological Carriers prepared fromco-stimulatory molecule transduced host cells would be HSV-1 & -2antibody reactive positive.

TABLE 2 HSV-1&-2 Specific Antibody Formation Induced by Exposure ofPeripheral Blood T-lymphocytes to HSV-2 based Biological CarrierPreparations Antibody Culture Reactivity Sample Time Point (relative ODunits) Sample From Against: Day 3 Day 6 Day 10 Day 14 ControlHSV-1 >4.00 >4.00 >4.00 >4.00 HSV-2 3.09 2.993 >4.00 2.688 HIV-1 2.232.187 2.698 1.889 VSV 0.26 0.159 0.245 0.029 Unstimulated HSV-1 0.0070.012 ND 0.001 HSV-2 0.006 0.004 0.002 HIV-1 0.003 0.007 0.004 VSV 0.0040.002 0.004 PHA-stimulated HSV-1 0.005 0.036 0.586 0.060 HSV-2 0.0050.015 0.590 0.020 HIV-1 0.007 0.009 0.005 0.003 VSV 0.004 0.002 0.0050.002 HSV-2 based HSV-1 0.214 1.876 3.722 0.006 BCs HSV-2 0.163 1.7443.503 0.003 HIV-1 0.003 0.004 0.005 0.003 VSV 0.005 0.003 0.003 0.007HIV-1 Based BC HSV-1 0.002 0.007 0.031 0.005 HSV-2 0.004 0.004 0.0270.002 HIV-1 0.004 0.001 0.005 0.001 VSV 0.003 0.002 0.007 0.004 BC =Biological Carrier; ND = not done

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

1. A non-infectious biological carrier comprising a HumanImmunodeficiency Virus (HIV) viral or viral-like particle that (i)presents at least one exogenous antigen incorporated into said particlesuch that said at least one antigen initiates an immune response; (ii)also expresses at least one exogenous antigen fragment bound to aprimary host cell surface molecule selected from MHC I and MHC II; and(iii) also expresses at least one B7 molecule as a co-stimulatorymolecule.
 2. The biological carrier of claim 1 wherein said at least oneB7 molecule is selected from B7-1 and B7-2.
 3. The biological carrier ofclaim 1 wherein the exogenous antigen is selected from the groupconsisting of a protein, a polypeptide, and an antibody.
 4. Thebiological carrier of claim 1 wherein said exogenous antigen isrecombinantly produced by a host cell.
 5. The biological carrier ofclaim 4 wherein the exogenous antigen is encoded in an expression vectorintroduced into the host cell.
 6. The biological carrier of claim 4wherein the exogenous antigen is from a pathogen.
 7. The biologicalcarrier of claim 4 wherein the exogenous antigen is from an infectiousagent.
 8. The biologically constructed particle of claim 4 wherein theexogenous antigen is from a tumor.
 9. The biologically constructedparticle of claim 8 wherein the tumor antigen is a tumor-specifictransplantation antigen.
 10. The biologically constructed particle ofclaim 8 wherein the tumor antigen is a tumor-associated transplantationantigen.
 11. The biological carrier of claim 1 wherein saidco-stimulatory molecule is recombinantly produced by a host cell. 12.The biological carrier of claim 6 wherein the exogenous antigen isencoded in an expression vector introduced into a host cell.
 13. Thebiological carrier of claim 1 which further contains a cytokinerecombinantly produced by a host cell.
 14. The biological carrier ofclaim 1 wherein said particle is derived from acute pathogen infectionof a host cell that has been modified to have at least one exogenousrecombinant molecule expressed on the host cell surface such that saidmolecule is incorporated into the particle as the particle is releasedfrom the host cell.
 15. The biological carrier of claim 14 wherein saidparticle is derived from a chronically pathogen infected host cell line.16. A method for inducing an immune response by stimulating presentationof at least one exogenous antigen, said method comprising administeringa biological carrier of claim 1 to an animal or human subject.